Using Bacillus subtilis as a gram-positive model, we propose to investigate the mechanism of proteolytic control that is applied in response to oxidative stress. Proteolytic control is essential for protein homeostasis and also plays a key role in modulating the levels of specific regulatory factors. One such factor, Spx, is a global transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Its concentration is elevated when cells undergo oxidative stress. When stress is alleviated, Spx is degraded by the ATP-dependent protease, ClpXP, a process that requires the substrate recognition factor, YjbH. Spx interacts directly with YjbH, which promotes ClpXP-catalyzed Spx proteolysis. Our recent studies show that a small peptide YirB binds to YjbH thereby releasing Spx from proteolytic control. One of our goals is to study the interactions between these four components, Spx/YjbH/ClpX/YirB, both in vivo and in vitro during oxidative stress and as stress subsides. This will be determined using active, affinity-tagged versions of the binding partners for pull down interaction experiments to be conducted with in vivo samples and in vitro reactions. In addition, to gaining in-depth understanding of the interactions, I will undertake structural studies of the key player YjbH and its complexes with Spx and YirB. During this award period, I intend to gain skills in bacterial genetics and knowledge to design approaches to elucidate the complexities of regulated proteolysis, while utilizing my advanced expertise in crystallography to define in detail the interactions between the protein components at the molecular level.